Summary. Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colonyforming assay in which samples from early passages were plated at low densities of about 10 cells per cm 2 . Samples with high colony-forming ef®ciency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.

Neurotransplantation has been used to explore the development of the central nervous system and for repair of diseased tissue in conditions such as Parkinson's disease. Here, we examine the effects of direct injection into rat brain of human marrow stromal cells (MSCs), a subset of cells from bone marrow that include stem-like precursors for nonhematopoietic tissues. Human MSCs isolated by their adherence to plastic were infused into the corpus striatum. Five to 72 days later, brain sections were examined for the presence of the donor cells. About 20% of the infused cells had engrafted. There was no evidence of an inflammatory response or rejection. The cells had migrated from the injection site along known pathways for migration of neural stem cells to successive layers of the brain. After infusion into the brain, the human MSCs lost their immunoreactivity to antibodies for collagen I. Initially, the human cells continued to stain with antibodies to fibronectin but the region of staining with fibronectin was significantly decreased at 30 and 72 days. The results suggest that MSCs may be useful vehicles for autotransplantation in both cell and gene therapy for a variety of diseases of the central nervous system.

Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF-1alpha, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF-1alpha were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF-1alpha subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF-1alpha protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF-1alpha protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF-1alpha transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF-1alpha is a metabolic adaptation of nucleus pulposus cells to a unique oxygen-limited microenvironment. The study confirmed that HIF-1alpha can be used as a phenotypic marker of nucleus pulposus cells.

Two overlapping cDNAs that encode a 197-kDa sequence-selective DNA-binding protein were isolated from libraries derived from mouse lymphoid cell mRNA. In addition to a DNA-binding domain, the protein contains both a chromodomain, which occurs in proteins that are implicated in chromatin compaction, and an SNF2/SWI2-like helicase domain, which occurs in proteins that are believed to activate transcription by counteracting the repressive effects of chromatin structure. A Southern blot analysis indicated that this protein, which we have named CHD-1, for chromodomainhelicase-DNA-binding protein, is present in most, if not all, mammalian species. A Northern blot analysis revealed multiple CHD mRNA components that differed both qualitatively and quantitatively among various cell types. The various mRNAs, which are probably produced by alternative RNA processing, could conceivably encode tissue-specific and developmental stage-specific isoforms of the protein. Based on its interesting combination of features, we suspect that CHD-1 plays an important role in gene regulation.In eukaryotic cells, selective transcription of discrete sets of genes is accomplished by a variety ofregulatory mechanisms. One major mechanism involves the concerted action of distinct combinations of proteins (transcription factors) that bind directly or indirectly to particular DNA sequences and act to stimulate or repress the basal transcriptional machinery (see ref. 1 for review). Another important mechanism regulates the accessibility of certain genes to the transcriptional apparatus by regional modifications of chromatin structure. Proteins participating in this type of mechanism could affect such processes as nucleosome packing, loop formation, DNA supercoiling, and attachment to the nuclear matrix (see ref Neither of these proteins has yet been found to possess DNA-binding capability.We report here on the isolation and characterization of cDNAs that encode a 197-kDa mouse protein with an SNF2 (Brm) pattern of helicase motifs in its central region and two other noteworthy features: a sequence-selective DNAbinding domain and a motif known as the chromodomain, which occurs in Pc and in HP1, a structural component of compacted chromatin (5, 21). The chromodomain is essential for the function of Pc, apparently being required for its assembly into chromatin as part of a multiprotein complex (5). Thus, the mouse protein, which we have termed CHD-1,t embodies within a single molecule characteristics of two functionally related proteins (Brm and Pc), as well as DNAbinding capability. This combination of features could endow CHD-1 with novel regulatory properties. MATERIALS AND METHODSIsolation of the KY3 and KY9 Clones and Plasmid Construction. Agtll phage from a mouse B-cell lymphoma (A20) cDNA expression library (Clontech) were screened with double-stranded oligonucleotide probes by the protocol of Vinson et al. (7) Isolation of Clone 3-3. A cDNA library was constructed from cytoplasmic poly(A)+ RNA of S194 plasmacytoma cells and the AZap...

CHD1 is a novel DNA-binding protein that contains both a chromatin organization modifier (chromo) domain and a helicase/ATPase domain. We show here that CHD1 preferentially binds to relatively long A ⅐ T tracts in double-stranded DNA via minor-groove interactions. Several CHD1-binding sites were found in a well-characterized nuclear-matrix attachment region, which is located adjacent to the intronic enhancer of the immunoglobulin gene. The DNA-binding activity of CHD1 was localized to a 229-amino-acid segment in the C-terminal portion of the protein, which contains sequence motifs that have previously been implicated in the minor-groove binding of other proteins. We also demonstrate that CHD1 is a constituent of bulk chromatin and that it can be extracted from nuclei with 0.6 M NaCl or with 2 mM EDTA after mild digestion with micrococcal nuclease. In contrast to another chromo-domain protein, HP1, CHD1 is not preferentially located in condensed centromeric heterochromatin, even though centromeric DNA is highly enriched in (A؉T)-rich tracts. Most interestingly, CHD1 is released into the cytoplasm when cells enter mitosis and is reincorporated into chromatin during telophase-cytokinesis. These observations lend credence to the idea that CHD1, like other proteins with chromo or helicase/ATPase domains, plays an important role in the determination of chromatin architecture.Previously, we reported on the discovery of a 196-kDa mouse protein with three signature motifs that relate it to families and superfamilies of other proteins (7). The three signature motifs are embodied in the name of this protein, CHD1, which stands for chromatin organization modifier (chromo)-helicase/ATPase-DNA-binding protein 1 (Fig. 1). One of the motifs, the 52-amino-acid chromo (C) domain, is present in proteins that have been implicated in the process of chromatin compaction. This process can occur over large genomic regions or at specific loci and can result in the repression of gene expression. Among the proteins with a C domain are the 25-kDa protein, HP1, which is present in the pericentric/constitutive heterochromatin of Drosophila, mouse, and human cells (17,31,32,37). Mutations in HP1 result in suppression of position-effect variegation, a process thought to involve the spreading of heterochromatin into euchromatic regions (9, 34). A C domain is also present in the 44-kDa Drosophila protein, Polycomb, and a possible mouse homolog, M33 (26, 27). Polycomb is involved in the repression of homeotic-gene expression, presumably by participating in the generation of heterochromatin at specific loci (24, 25). Thus, mutations in polycomb result in the ectopic expression of homeotic genes.The helicase/ATPase (H) domain represents a second signature motif of CHD1. This domain is present in a large superfamily of proteins with diverse functions, such as the replication, recombination, and repair of DNA and the transcription, processing, and translation of RNA. The H domain of CHD1 is most closely related to that of the yeast proteins SWI2/SNF2...

Human collagen alpha 3(VI) chain mRNA (approximately 10 kb) was cloned and shown by sequence analysis to encode a 25 residue signal peptide, a large N‐terminal globule (1804 residues), a central triple helical segment (336 residues) and a C‐terminal globule (803 residues). Some of the sequence was confirmed by Edman degradation of peptides. The N‐terminal globular segment consists of nine consecutive 200 residue repeats (N1 to N9) showing internal homology and also significant identity (17‐25%) to the A domains of von Willebrand Factor and similar domains present in some other proteins. Deletions were found in the N3 and N9 domains of several cDNA clones suggesting variation of these structures by alternative splicing. The C‐terminal globule starts immediately after the triple helical segment with two domains C1 (184 residues) and C2 (248 residues) being similar to the N domains. They are followed by a proline rich, repetitive segment C3 of 122 residues, with similarity to some salivary proteins, and domain C4 (89 residues), which is similar to the type III repeats present in fibronectin and tenascin. The most C‐terminal domain C5 (70 residues) shows 40‐50% identity to a variety of serine protease inhibitors of the Kunitz type. The whole sequence contains 29 cysteines which are mainly clustered in short segments connecting domains N1, C1, C2 and the triple helix, and in the inhibitor domain. Five putative Arg‐Gly‐Asp cell‐binding sequences are exclusively localized in the triple helical segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective. To study the changes in patterns of gene expression exhibited by human chondrocytes as they dedifferentiate into fibroblastic cells in culture in order to better understand the mechanisms that control this process and its relationship to the phenotypic changes that occur in chondrocytes during the development of osteoarthritis (OA).Methods. Human fetal epiphyseal chondrocytes (HFCs) were cultured either on poly-(2-hydroxyethyl methacrylate)-coated plates (differentiated HFC cultures) or in plastic tissue culture flasks as monolayers (dedifferentiated HFC cultures). Following 11 days of culture under either condition, poly(A؉) RNA was isolated from the two cell populations and subjected to a gene expression analysis using a microarray containing ϳ5,000 known human genes and ϳ3,000 expressed sequence tags (ESTs).Results. A >2-fold difference in the expression of 62 known genes and 6 ESTs was observed between the two cell types. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Northern analyses. Two transcription factor genes, TWIST and HIF-1␣, and a cellular adhesion protein gene, cadherin 11, were markedly regulated in response to differentiation and dedifferentiation. Expression of these genes was also detected in adult normal and OA cartilage and chondrocytes. Analysis of the gene expression profile of HFCs revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes.Conclusion. Chondrocytes in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one of expression of chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. Changes in gene expression associated with the process of dedifferentiation of HFCs in vitro were observed in a wide variety of genes, including genes encoding extracellular matrix proteins, transcription factors, and growth factors. At least 3 of the genes that were regulated in response to dedifferentiation were also found to be expressed in adult normal and OA articular cartilage and chondrocytes.

CHD1, an Mr approximately 200,000 protein that contains a chromodomain (C), an ATPase/helicase-like domain (H) and a DNA-binding domain (D), was previously shown to be associated with decompacted interphase chromatin in mammalian cells and with transcriptionally active puffs and interbands in Drosophila polytene chromosomes. We now show by transient transfection experiments with genes expressing wild-type and mutant forms of CHD1 that both the C and H domains are essential for its proper association with chromatin. We also present evidence for an in vivo interaction between CHD1 and a novel HMG box-containing protein, SSRP1, which involves an amino-terminal segment of CHD1 that does not include the chromodomain. Immunocytochemical analyses indicated that CHD1 and SSRP1 colocalize in both mammalian nuclei and Drosophila polytene chromosomes.